1. Field of Invention
The present invention relates to the field of Real time PCR. In particular, the present invention is directed to a system for performing multiplex real time PCR.
2. Prior Art Background
Amplification of DNA by polymerase chain reaction (PCR) is a technique fundamental to molecular biology. Nucleic acid analysis by PCR requires sample preparation, amplification, and product analysis. Although these steps are usually performed sequentially, amplification and analysis can occur simultaneously. DNA dyes or fluorescent probes can be added to the PCR mixture before amplification and used to analyze PCR products during amplification. Sample analysis occurs concurrently with amplification in the same tube within the same instrument. This combined approach decreases sample handling, saves time, and greatly reduces the risk of product contamination for subsequent reactions, as there is no need to remove the samples from their closed containers for further analysis. The concept of combining amplification with product analysis has become known as “real time” PCR. See, for example, U.S. Pat. No. 6,174,670.
Monitoring fluorescence during each cycle of PCR initially involved the use of ethidium bromide. (Higuchi R, G Dollinger, P S Walsh and R. Griffith, Simultaneous amplification and detection of specific DNA sequences, Bio/Technology 10 (1992) 413-417; Higuchi R, C Fockler G Dollinger and R Watson, Kinetic PCR analysis: real time monitoring of DNA amplification reactions, Bio/Technology 11 (1993) 1026-1030). In that system fluorescence is measured once per cycle as a relative measure of product concentration. Ethidium bromide detects double stranded DNA; if template is present fluorescence intensity increases with temperature cycling. Furthermore, the cycle number where an increase in fluorescence is first detected increases inversely proportionally to the log of the initial template concentration. Other fluorescent systems have been developed that are capable of providing additional data concerning the nucleic acid concentration and sequence.
In kinetic real time PCR, the formation of PCR products is monitored in each cycle of the PCR. The amplification is usually measured in thermocyclers which have additional devices for measuring fluorescence signals during the amplification reaction.
Real Time PCR Instrumentation
Several types of real time detection thermocyclers are known in the art.
EP 0 640 828, for example discloses an apparatus for monitoring multiple nucleic acid amplification simultaneously. It is characterized in that it comprises a metal block thermal cycler, including a heat conducting member having multiple recesses formed therein in order to take up a multiwell plate such as a microtiter plate. Detection is obtained by means of a CCD camera, arranged for detecting light emitted (simultaneously) from all of said recesses. Alternatively, the use of fiber optics is suggested. Depending on the fluorescent dyes which are used and—more important—, depending on the presence of a filter wheel close to the CCD camera, the system has the capability of analyzing multiplex amplification reactions, wherein in one reaction chamber, one or more different amplicons are detected by 2 or more differently labeled hybridization probes. Yet, EP 0 640 828 does neither anticipate nor suggest, which labels or detection formates could be used for such a multiplex/multicolor approach.
U.S. Pat. No. 6,015,674 discloses an apparatus and a system for real time PCR detection and quantification, characterized in that it is capable of detecting first and second fluorescent indicators, which may be used as labels for different hybridization probes in order to detect different amplicons in the same reaction vessel. Yet, U.S. Pat. No. 6,015,674 does not disclose a system for performing multiple experiments with a higher degree of complexity.
Another typical example is the Roche Diagnostics LightCycler (Cat. No. 2 0110468). It is a fast PCR system enabling kinetic on-line PCR quantification and subsequent analysis of PCR-product melting curves. The optical system of the current LightCycler version 1.2 being commercially available contains one light source, a blue light emitting diode (470 nm LED) and three detection channels. The amplification products are detected by means of fluorescent labeled hybridization probes which only emit fluorescence signals when they are bound to the target nucleic acid or in certain cases also by means of fluorescent dyes that bind to double-stranded DNA. A defined signal threshold is determined for all reactions to be analysed and the number of cycles Cp required to reach this threshold value is determined for the target nucleic acid as well as for the reference nucleic acids such as the standard or housekeeping gene. The absolute or relative copy numbers of the target molecule can be determined on the basis of the Cp values obtained for the target nucleic acid and the reference nucleic acid.
The fluorescence emitted by a sample is separated by a set of dichroic mirrors and filters into different wavelengths that can be recorded in one of the three detection channels (530/640/710 nm). This allows detection of the double-stranded DNA-binding dye SybrGreenI, mono color detection of the TaqMan Probe format and dual color detection of the Hybridization Probe (HybProbe) format. Details of the Lightcycler system are disclosed in WO 97/46707, WO 97/46712 and WO 98/46714, The complete contents of these applications are herewith incorporated by reference.
A very important feature of the LightCycler instrument is the color compensation software. In principle this software allows for accurate quantification and melting curve analysis by means of correcting spectral overlap of monitored fluorescent radiation in a temperature dependent manner. Technical details are disclosed in U.S. Pat. No. 6,197,520.
Similar to the LightCycler system, the Corbett Rotor-Gene Real time PCR Thermocycler (www.corbettresearch.com) is a 4 channel multiplexing system comprising 4 different LEDs as excitation sources and corresponding photodiodes as fluorescent detection units. Thus, although this instrument hardware at least theoretically has the capacity of performing multiplex experiments with up to four differently labeled hybridization probes within one reaction vessel, no respective successful application protocol has been published so far.
Another real time PCR instrument is the Biorad iQ Multi-color Real time PCR detection system (Cat. No: 170-8740), which allows for a fluorophore excitation and emission from 400 nm to 700 nm. The system is based on a conventional multiwell heating block for thermocycling, a tungsten lamp as an excitation source, a filter wheel for providing appropriate excitation wavelengths, a second filter wheel for selecting appropriate emission wavelengths and a CCD camera as a detection unit. The instrument has successfully been used in a multiplex assay for the detection of 4 different amplicons generated from targets with more or less equimolar concentrations, using four differently labeled TaqMan probes in the same reaction vessel (Pedersen, S., Bioradiations 107 (2001) 10-11).
In a further approach to increase multiplexing capacities, U.S. Pat. No. 6,369,893 discloses a real time PCR thermocycling instrument comprising a first optics assembly with at least two light sources and a second optics assembly comprising at least two detectors for detecting and discriminating light of different emission wavelengths. In particular, a specific embodiment of different 4 LEDs as light sources and 4 different photodiodes as detectors is disclosed. Thus, the instrument disclosed in U.S. Pat. No. 6,369,893 in principle can be used for real time PCR detection with a broad selection of different fluorescent dyes which are known in the art. Yet, U.S. Pat. No. 6,369,893 does neither anticipate nor suggest any approach on how a multiplex experiment comprising multiple different probes each labeled with a different fluorescent entity needs to be designed.
Real Time PCR Detection Formates
In general, there exist different formates for real time detection of amplified DNA, of which the following are well known and commonly used in the art:
a) DNA Binding Dye Formate
Since the amount of double stranded amplification product usually exceeds the amount of nucleic acid originally present in the sample to be analyzed, double-stranded DNA specific dyes may be used, which upon excitation with an appropriate wavelength show enhanced fluorescence only if they are bound to double-stranded DNA. Preferably, only those dyes may be used which like SybrGreenI, for example, do not affect the efficiency of the PCR reaction.
All other formates known in the art require the design of a fluorescent labeled Hybridization Probe which only emits fluorescence upon binding to its target nucleic acid.
b) TaqMan Probe
A single-stranded Hybridization Probe is labeled with two components. When the first component is excited with light of a suitable wavelength, the absorbed energy is transferred to the second component, the so-called quencher, according to the principle of fluorescence resonance energy transfer. During the annealing step of the PCR reaction, the hybridization probe binds to the target DNA and is degraded by the 5′-3′ exonudease activity of the Taq Polymerase during the subsequent elongation phase. As a result the excited fluorescent component and the quencher are spatially separated from one another and thus a fluorescence emission of the first component can be measured (U.S. Pat. No. 5,538,848).
c) Molecular Beacons
These hybridization probes are also labeled with a first component and with a quencher, the labels preferably being located at both ends of the probe. As a result of the secondary structure of the probe, both components are in spatial vicinity in solution. After hybridization to the target nucleic acids both components are separated from one another such that after excitation with light of a suitable wavelength the fluorescence emission of the first component can be measured (U.S. Pat. No. 5,118,801).
d) Single Label Probe (SLP) Format
This detection format consists of a single oligonucleotide labeled with a single fluorescent dye at either the 5′- or 3′-end (WO 02/14555). Two different designs can be used for oligo labeling: G-Quenching Probes and Nitroindole-Dequenching probes.
In the G-Quenching embodiment, the fluorescent dye is attached to a C at oligo 5′- or 3′-end. Fluorescence decreases significantly when the probe is hybridized to the target, in case two G's are located on the target strand opposite to C and in position 1 aside of complementary oligonucleotide probe.
In the Nitroindole Dequenching embodiment, the fluorescent dye is attached to Nitroindole at the 5′- or 3′-end of the oligonucleotide. Nitroindole somehow decreases the fluorescent signaling of the free probe. Fluorescence increases when the probe is hybridized to the target DNA due to a dequenching effect.
e) FRET Hybridization Probes
The FRET Hybridization Probe test format is especially useful for all kinds of homogenous hybridization assays (Matthews, J. A., and Kricka, L. J., Analytical Biochemistry 169 (1988) 1-25. It is characterized by a pair of two single-stranded hybridization probes which are used simultaneously and are complementary to adjacent sites of the same strand of the amplified target nucleic acid. Both probes are labeled with different fluorescent components. When excited with light of a suitable wavelength, a first component transfers the absorbed energy to the second component according to the principle of fluorescence resonance energy transfer such that a fluorescence emission of the second component can be measured when both hybridization probes bind to adjacent positions of the target molecule to be detected.
When annealed to the target sequence, the hybridization probes must sit very close to each other, in a head to tail arrangement. Usually, the gap between the labeled 3′ end of the first probe and the labeled 5′ end or the second probe is as small as possible, i.e. 1-5 bases. This allows for a close vicinity of the FRET donor compound and the FRET acceptor compound, which is typically 10-100 Angstroem.
Alternatively to monitoring the increase in fluorescence of the FRET acceptor component, it is also possible to monitor fluorescence decrease of the FRET donor component as a quantitative measurement of hybridization event.
In particular, the FRET Hybridization Probe format may be used in real time PCR, in order to detect the amplified target DNA. Among all detection formats known in the art of real time PCR, the FRET-Hybridization Probeformat has been proven to be highly sensitive, exact and reliable (WO 97/46707; WO 97/46712; WO 97/46714). Yet, the design of appropriate FRET Hybridization Probe sequences may sometimes be limited by the special characteristics of the target nucleic acid sequence to be detected.
As an alternative to the usage of two FRET hybridization probes, it is also possible to use a fluorescent-labeled primer and only one labeled oligonucleotide probe (Bernard, P. S., et al., Analytical Biochemistry 255 (1998) 101-107). In this regard, it may be chosen arbitrarily, whether the primer is labeled with the FRET donor or the FRET acceptor compound.
Besides PCR and real time PCR, FRET hybridization probes are used for melting curve analysis. In such an assay, the target nucleic acid is amplified first in a typical PCR reaction with suitable amplification primers. The hybridization probes may already be present during the amplification reaction or added subsequently. After completion of the PCR-reaction, the temperature of the sample is constitutively increased, and fluorescence is detected as long as the hybridization probe was bound to the target DNA. At melting temperature, the hybridization probes are released from their target, and the fluorescent signal is decreasing immediately down to the background level. This decrease is monitored with an appropriate fluorescence versus temperature-time plot such that a first derivative value can be determined, at which the maximum of fluorescence decrease is observed.
There exist many different pairs of fluorescent dyes known in the art which according to the invention are principally capable of acting together as a FRET donor/FRET acceptor pair. Yet, prior to the present invention, no functional example has been disclosed, characterized in that 4 different FRET pairs have succesfully been used in a multiplex detection assay. Among other reasons, this may be due to lack of approriate instrumentation and, moreover, due to fact that the functionality of the FRET process of a specific FRET pair is interfered by other fluorecent compounds which are present in the same reaction mixture.
As discussed above, there exist different real time detection thermocycler instruments having a maximum of 4 detector channels for multiplex/multicolor detection. Yet, the utility of all of these instruments for multiplex detection up to now has been very limited due to the fact that attempts to establish real time multicolor multiplex assays with several (at least more than two) differently labeled probes with sufficient sensitivity and specificity have not been successful so far.
Thus, it was the object of the present invention to provide an improved system which allows for an optimized and at the same time flexible design of multiplex/multicolor detection experiments. In one aspect the problem to be solved relates to improvements in the design of approriate hybridization probes. In another aspect, the problem to be solved relates to an improvement of instrumentation.